This invention relates to fluorescent intensity based methods for assaying binding between ligands and receptors, particularly ligands and receptors comprising amino acids and/or amino acid analogs.
A detection method typically employs at least one analytical reagent that binds to a specific target macromolecular species and produces a detectable signal. These analytical reagents typically have two components: (1) a probe macromolecule, for example, an antibody or oligonucleotide, that can bind a target macromolecule with a high degree of specificity and affinity, and (2) a detectable label, such as a radioisotope or covalently-linked fluorescent dye molecule. In general, the binding properties of the probe macromolecule define the specificity of the detection method, and the detectability of the associated label determines the sensitivity of the detection method. The sensitivity of detection is in turn related to both the type of label employed and the quality and type of equipment available to detect it.
For example, radioimmunoassays have been among the most sensitive and specific analytical methods used for detecting and quantitating biological macromolecules. Radioimmunoassay techniques have been used to detect and measure minute quantities of specific analytes, such as polypeptides, drugs, steroid hormones, polynucleotides, metabolites, and tumor markers, in biological samples. Radioimmunoassay methods employ immunoglobulins labeled with one or more radioisotopes as the analytical reagent. Radiation (alpha, beta or gamma) produced by decay of the attached radioisotope label serves as the signal which can be detected and quantitated by various radiometric methods.
Radioisotope based assays used to measure peptide-protein, ligand-receptor or drug-receptor interactions have typically required one component (usually the peptide, ligand or drug) to be radioactively labeled with either 125I, 35S, 32P or 3H for detection and quantitation. Radioactive labeling is labor-intensive, time-consuming, a health and environmental hazard during synthesis, purification, storage, usage and disposal steps, and is relatively expensive. Radioligands or radioactively labeled peptides are most commonly labeled with 125I, which emits mainly xcex3 radiation and some xcex2 radiation, and has a half-life of sixty days. 125I is considered more dangerous than the other above-mentioned radioisotopes, due to its high volatility and the fact that it concentrates in the thyroid, thereby requiring more precautions and expense during its handling.
Conventionally, it has been necessary to separate interacting complexes from unbound radioactive probes either by filtration and extensive washing steps or by native polyacrylamide gel electrophoresis, followed by gel drying. Low affinity binding interactions may dissociate during extensive washing steps or migration through a gel, and thus not be detected. Dried gels are analyzed by autoradiography or phosphoimaging, which is very time-consuming, especially when 35S or 3H isotopes are used. Filtered 125I-bound complexes are analyzed by scintillation counting in special xcex3 radiation scintillation counters.
Moreover, the sensitivity of traditional detection methods, such as radioimmunoassays, can be too low to detect particularly small or dilute samples.
Thus, a need has existed in the art for a simple, highly sensitive, effective and rapid method for analyzing interaction between ligands and receptors, particularly ligands and receptors comprising amino acids and/or amino acid analogs.
All references cited herein are incorporated herein by reference in their entireties.
The invention provides a method for assaying binding between at least one target and at least one probe, said method comprising:
providing at least one target comprising a peptide sequence or a peptide analog sequence;
providing at least one probe comprising an amino acid or a second amino acid analog, and at least one fluorophore;
providing a test medium comprising said at least one target and said at least one probe;
irradiating said test medium with radiation effective to cause said at least one fluorophore to emit fluorescent light; and
detecting a quenching effect on fluorescence emitted by said at least one fluorophore resulting from said binding,
wherein said binding is specific, and wherein said method is conducted without separating complexes of said at least one target and said at least one probe from free target and free probe prior to said quenching effect detecting, and without providing a signal quenching agent to quench said emitted fluorescence.